Project Summary Our recent work has shown first that endogenous hydrogen sulfide (H2S)-derived from cystathionine beta-synthase (CBS) functions as a novel vasodilator system for mediating pregnancy-augmented and agonist-stimulated uterine vasodilation as measured by a rise in uterine blood flow that is rate-limiting for pregnancy health. However, it is completely unknown how H2S exerts its vasodilatory effect in the uterine artery (UA). S-sulfhydration (formation of -S-SH adducts from cysteines in proteins) has gained significant momentum in the last few years as the primary signaling mechanism for mediating H2S action. In this R21 we will test a novel hypothesis that an unbiased quantitative proteomics method based on tag-switch, stable-isotope labeling by amino acids in cell culture (SILAC), and mass spectrometry (MS) can be developed to determine if pregnancy augments cell (endothelial vs. smooth muscle)- specific UA protein S-sulfhydration via a CBS-H2S pathway. To address this hypothesis, we will develop a comprehensive but simple quantitative proteomics method for determining sulfhydrated proteins in the proteomes of human UA endothelial cells (hUAEC) and smooth muscle cells (hUASMC) isolated from nonpregnant (NP) vs. pregnant (P) women, named as NP- and P- hUAEC or hUASMC, respectively. We will also analyze the sulfhydrated proteins in UAs isolated from hysterectomies of age-matched NP vs. P women who give birth of boys and girls. We believe that the proposed studies are highly exploratory and with high risk because neither the method to be developed nor pregnancy-dependent sulfhydrated UA protein networks have been established. This project is also a new direction in the PI?s laboratory and thus best fits the R21 funding mechanism. Nonetheless, the potential reward certainly offsets the risk because the outcomes will significantly move uterine hemodynamics biology forward. The new proteomics method to be developed can identify all sulfhydrated proteins in paired proteomes with unbiased quantitation and simultaneous identification of specific sulfhydration site(s) in each sulfhydrated protein. It can be applied for analyzing global protein sulfhydration in any biological settings to facilitate the understanding of the mechanisms of H2S action. We believe that quantitative comparisons of sulfhydrated proteomes in NP vs. P hUAEC and hUASMC and NP vs. P human UA are critically important because data obtained will provide fundamental sulfhydrated protein networks required for defining the biological and clinical significance of protein sulfhydration in human UAEC and UASMC. Since the data of these studies are generated from human UA and primary cells derived from these samples, the results of these studies will be translational. The impact of this work is that once pregnancy-dependent sulfhydrated protein networks are identified in human UAEC and UASMC, we can use this knowledge to determine the physiological and clinical significance of sulfhydration of target proteins via enhanced CBS/H2S pathway in pregnancy-associated and agonist-stimulated rises in uterine blood flow crucial for pregnancy health.